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2025年8月3日 星期日
农业生物技术学报  2022, Vol. 30 Issue (8): 1640-1648    DOI: 10.3969/j.issn.1674-7968.2022.08.019
  研究资源与技术改进 本期目录 | 过刊浏览 | 高级检索 |
黄瓜绿斑驳花叶病毒SYBR GreenⅠ实时荧光RT-PCR检测方法的建立
田沂民1, 吴建祥2, 陈磊3, 罗金燕3, 滕凯4, 叶军1, 杜伟5, 于翠1,*
1 上海海关 动植物与食品检验检疫技术中心,上海 200135;
2 浙江大学 农业与生物技术学院,杭州 310058;
3 上海农业技术推广服务中心,上海 201103;
4 上海海关 动植物检疫监管处,上海 200082;
5 宁夏回族自治区农业技术推广总站, 银川 750001
Development of SYBR GreenⅠReal-time RT-PCR for the Detection of Cucumber green mottle mosaic virus
TIAN Yi-Min1, WU Jian-Xiang2, CHEN Lei3, LUO Jin-Yan3, TENG Kai4, YE Jun1, DU Wei5, YU Cui1,*
1 Technical Center for Animal, Plant and Food Inspection and Quarantine of Shanghai Customs District, Shanghai 200135, China;
2 College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China;
3 Shanghai Agricultural Technology Extension Center, Shanghai 201103, China;
4 Division of Animal and Plant Quarantine and Inspection of Shanghai Customs District, Shanghai 200082, China;
5 Agricultural Technology Extension Station of Ningxia, Yinchuan 750001, China
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摘要 黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus, CGMMV)是我国重要的检疫性植物种传病毒,在世界各地均有发生,造成西瓜(Citrullus lanatus)、黄瓜(Cucumis sativus)、甜瓜(Cucumis melo)等作物的严重经济损失。CGMMV可由种子直接传播至下一代秧苗,为保障种子贸易顺利进行,有必要建立快速而准确的病毒检测方法。本研究以截获的带毒种子为材料,针对CGMMV建立了SYBR GreenⅠ实时荧光RT-PCR检测方法,其中设计筛选特异、灵敏的引物是关键。利用多次筛选得到的引物对建立了SYBR GreenⅠ实时荧光RT-PCR检测方法,实验结果表明该方法能够特异地检测CGMMV,不与其他侵染葫芦科植物的病毒发生交叉反应;灵敏度是RT-PCR方法的1 000倍,是TaqMan探针实时荧光RT-PCR方法的100倍;且整个检测过程可在2.5 h内完成,是一种快速、灵敏、可靠的检测方法;应用该方法及TaqMan实时荧光RT-PCR方法同时对国内外198份种苗样品进行检测,结果显示kappa值大于0.82,说明两种方法具有高度一致性,本研究建立的SYBR GreenⅠ实时荧光RT-PCR方法有效、可靠,为种子快速通关提供新的技术手段。
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田沂民
吴建祥
陈磊
罗金燕
滕凯
叶军
杜伟
于翠
关键词 黄瓜绿斑驳花叶病毒(CGMMV)SYBR GreenⅠ实时荧光RT-PCR种子灵敏度特异性    
AbstractCucumber green mottle mosaic virus (CGMMV) is an important quarantine plant virus and is also transmitted by seeds which often causes seriously damages to productions of watermelon (Citrullus lanatus), melon (Cucumis melo), cucumber (Cucumis sativus), and other cucurbits. To guarantee the smooth proceeding of seed trade, it is necessary to establish more rapidly and precisely detection methods. This study developed a SYBR GreenⅠreal-time RT-PCR technique for detection of CGMMV in seeds. It is crucial to design and screen a pair of specific primers based on the conserved CGMMV gene sequences. The result showed that the development of SYBR GreenⅠreal time RT-PCR method in this study could detect CGMMV specially, and there was no cross-reactivity with other common cucurbits viruses. Sensitivity assay showed that the sensitivity of this method was 1 000 times that of RT-PCR and 100 times that of TaqMan RT-PCR. And the whole detection process could be completed within 2.5 h, indicating that the SYBR GreenⅠreal-time RT-PCR was a rapid, sensitive and simple detection method. Cucurbitaceae seeds, seedlings, and fruits imported and purchased from domestic market were employed to be detected by SYBR GreenⅠreal-time RT-PCR and TaqMan real-time RT-PCR simultaneously. The result showed that the value of kappa of the two method was above 0.82, indicating that the novel SYBR GreenⅠreal-time RT-PCR method could be effective and reliable. The present study provides new technical means for rapid clearance of seeds.
Key wordsCucumber green mottle mosaic virus (CGMMV)    SYBR GreenⅠreal-time RT-PCR    Seed    Sensitivity    Specificity
收稿日期: 2021-09-30     
ZTFLH:  S41-30  
基金资助:上海市科技兴农项目(2019-02-08-00-08-F01150); 宁夏回族自治区重点研发计划(2020BBF03004)
通讯作者: *yucuicn@163.com   
引用本文:   
田沂民, 吴建祥, 陈磊, 罗金燕, 滕凯, 叶军, 杜伟, 于翠. 黄瓜绿斑驳花叶病毒SYBR GreenⅠ实时荧光RT-PCR检测方法的建立[J]. 农业生物技术学报, 2022, 30(8): 1640-1648.
TIAN Yi-Min, WU Jian-Xiang, CHEN Lei, LUO Jin-Yan, TENG Kai, YE Jun, DU Wei, YU Cui. Development of SYBR GreenⅠReal-time RT-PCR for the Detection of Cucumber green mottle mosaic virus. 农业生物技术学报, 2022, 30(8): 1640-1648.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2022.08.019     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2022/V30/I8/1640
 
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